AMPO spin traps

ABSTRACT

Provided are spin traps for the study of radical formation in vivo or in vitro. 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO) and 2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH 2 -AMPO), have the following structures, respectively:  
                 
as well as salts thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 60/630,418 entitled AMPO SPIN TRAPS and filed Nov. 23, 2004, theentirety of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Spin traps were originally used to measure free radical activity becausethey are able to react with free radicals both in vitro and in vivo andcan be measured by a number of different techniques, including ESR andNMR. Originally used to measure the efficacy of other anti-oxidants,spin traps have since been recognized that spin traps themselves may bean important tool in treating a variety of conditions, includinginflammatory and degenerative age-related diseases.

SUMMARY OF THE INVENTION

Provided are 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO) and2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH₂-AMPO), which havethe following structures, respectively:

as well as salts thereof. In some embodiments, the methyl group may bereplaced by another group, such as halo or substituted or unsubstitutedstraight, branched or cyclic alkyl, provided that the compound is stillsuitable as a spin trap.

The compounds described herein are useful to study radical formation,including but not limited to hydroxyl, superoxide, C-centered, sulfite,and tert-butoxyl radicals. The compounds described herein areparticularly useful for studying radical formation in aqueous solutionsboth in vitro and in vivo. In some embodiments, radicals may be detectedby ESR spectroscopy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. 400 MHz ¹H-NMR spectrum of AMPO in CDCl₃.

FIG. 2.400 MHz ¹H-NMR spectrum of AMPO in D₂O.

FIG. 3. 100 MHz ¹³C-NMR spectrum of AMPO in CDCl₃.

FIG. 4. Neat FT-IR spectrum of AMPO.

FIG. 5. ESI-Mass Spectrum of AMPO.

FIG. 6. 400 MHz ¹H-NMR spectrum of NH₂-AMPO in D₂O.

FIG. 7. Neat FT-IR spectrum of NH₂-AMPO.

FIG. 8. ESI-Mass Spectrum of NH₂-AMPO. The (C₆H₁₁N₃O₂H)⁺ peak has adifference of +6.6 ppm compared to the exact mass, while that of(C₆H₁₁N₃O₂Na)⁺ has <−1.0 ppm difference.

FIG. 9. UV-Vis spectrum of 0, 60 and 90 μM NH₂-AMPO.

FIG. 10. High performance liquid chromatogram of NH₂-AMPO. Condition:Stationary phase: C18 column (4.6 mm×25 cm) with particle size of 5 μm;flow rate=1.2 mL/min; Solvent: 50:50 (acetonitrile/phosphate-buffer pH7.4); Detector: UV 230 nm (black line) and 270 nm (green line). Inset:Total integration of all the peaks showing about 2% of 230 nm absorbingimpurities.

FIG. 11. GC-MS chromatogram (top) and spectrum (bottom) of NH₂-AMPO. Thecompound NH₂-AMPO has retention time of 16.06 min with a molecular ionpeak of 157 m/z.

FIG. 12.400 MHz 1H-NMR spectrum of EMPO in D₂O.

FIG. 13. Experimental (left) and simulated (right) EPR spectra of AMPOradical adducts with (a) .OH; (b) CO₂ ^(•−); (c) GS.; (d) SO₃ ^(•−); (e)tert-BuO. and (f) CH₃.CHOH. See experimental methods of radicalgeneration and spectrometer settings.

FIG. 14. EPR spectral profile of AMPO-O₂H (a) generated byxanthine-xanthine oxidase; (b) simulated spectrum based on theparameters described in Table 2; (c) generated by light-riboflavinsystem (note the significant contribution from a C-centered adduct). Seeexperimental methods for spectrometer settings. Arrow indicates the peakbeing monitored during kinetic studies.

FIG. 15. Superoxide adduct formation by 25 mM AMPO using PMA-activatedneutrophiles. (Top to bottom): 2, 10, 30, 60 min after the addition ofPMA.

FIG. 16. A view of the X-ray structure of AMPO. The non-hydrogen atomsare drawn with 50% probability displacement ellipsoids. The hydrogenatoms are drawn with an arbitrary radius.

FIG. 17. A view of the X-ray structure of NH₂-AMPO. The non-hydrogenatoms are drawn with 50% probability displacement ellipsoids. Thehydrogen atoms are drawn with an arbitrary radius.

DETAILED DESCRIPTION OF THE INVENTION

Provided are new spin traps and methods of making and using the same.The spin traps described herein include the nitrone5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO), the amido nitronecompound 2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH₂-AMPO) andderivatives thereof. Spin trapping by AMPO of hydroxyl, superoxide,C-centered, sulfite, and tert-butoxyl radicals has been demonstrated byelectron paramagnetic resonance (EPR) spectroscopy, making thesecompounds useful for the study of radical production in aqueous systems.

The nitrone 5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO) wassuccessfully synthesized and characterized. Spin trapping by AMPO ofhydroxyl, superoxide, C-centered, sulfite, and tert-butoxyl radicals hasbeen demonstrated for the first time by electron paramagnetic resonance(EPR) spectroscopy. Resulting spin adducts for each of these radicalsgave unique spectral profiles. Rate of superoxide radical trapping wasobtained by competitive trapping by AMPO versus DEPMPO and gave kAMPO=38M⁻¹ s⁻¹ (based on kDEPMPO=58 M⁻¹ s⁻¹) comparable to that of EMPOkEMPO=44 M⁻¹ s⁻¹. The half-life of AMPO-O₂ adduct is about t_(1/2)˜10minutes similar to that observed from EMPO but significantly shorterthan that of DEPMPO-O₂ adduct t_(1/2)˜16 minutes. Theoretical analysesusing density functional theory calculations at theB3LYP/6-31=G**/B3LYP6-31G* level were performed on AMPO and itscorresponding suproxide product. Calculations predicted the presence ofintramolecular H-bonding in both AMPO and its superoxide adduct, andthese interactions were further confirmed by an X-ray structure (in thecase of AMPO) of a novel and the amido nitrone compound2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH₂-AMPO). Thethermodynamic quantities for superoxide radical trapping by variousnitrones have been found to predict favorable formation of certainisomers. The measured partition coefficient in an n-octanol/buffersystem of AMPO gave a comparable value to those of DMPO and DEPMPO. Thisstudy demonstrates the suitability of AMPO nitrone as spin trap to studyradical production in aqueous systems.

These new compounds are useful both to study radical production inaqueous systems, both in vitro and in vivo. Additionally, thesecompounds may be useful in the treatment of inflammatory conditions andchronic degenerative diseases of aging. Some conditions that thesecompounds may be useful in treating include but are not limited to AIDS,arthritis, arteriosclerosis, Alzheimer's disease and otherpro-inflammatory disease conditions.

General Experimental Procedure for the Preparation of AMPO and NH₂-AMPOAll chemicals were purchased and used without further purification.Elemental analysis was performed by a commercial analytical servicecompany. ¹H-NMR and ¹³C-NMR measurements were performed on a 400 MHz and100 MHz spectrometer. FT-IR measurements were performed using neatsamples.

5-Ethoxycarbonyl-5-methyl-1-pyrroline N-oxide (EMPO). EMPO wassynthesized according to the method described previously by Bonnett, etal.¹ EMPO: clear liquid; ¹H NMR (400 MHz, D₂O) δ 1.23 (3 H, t, O—CH₂),1.61 (3 H, s, C(5)Me), 2.23-2.28 and 2.56-2.63 (2 H, m, C(4)H),2.76-2.81 (2 H, m, C(3)H), 4.19-4.25 (2H, q, O—CH₂CH₃), 7.37 (1H, t,C(2)H). IR (Neat film) 1737 (C═O), 1583 (C═N), 1214 (N—O).

5-Carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO). AMPO was prepared fromEMPO based on the procedure described previously with minormodification.² A solution of 0.5 g of EMPO was mixed with 10 mL ofconcentrated ammonium hydroxide in a sealed tube for 5 days at roomtemperature with shaking. The mixture was rotary evaporated to yield aviscous dark oil and passed through a silica gel column (200-400 mesh 60Å) twice using methanol-ethyl acetate (30:70) as solvent. Whitecrystalline product was obtained (0.10 g, 24%), mp. 134-135° C. (lit.137° C.)., (corrected using 3,4 dimethoxybenzoic acid, m.p., 180° C. andurea, m.p., 135° C.). ¹H NMR (400 MHz, D₂O) δ 1.64 (3 H, s, C(5)Me),2.21-2.29 and 2.54-2.61 (2 H, m, C(4)H), 2.73-2.73 (2 H, m, C(3)H), 7.36(1 H, t, C(2)H). ¹H NMR (400 MHz, CDCl₃) 1.90 (3 H, s, C(5)Me),2.27-2.34 and 3.14-3.20 (2 H, m, C(4)H), 2.79 (2 H, m, C(3)H), 7.19 (1H, t, C(2)H), 5.85 and 8.46 (2 H, br, NH₂). ¹³C NMR (100 MHz, CDCl₃) δ24.2 (s, —CH₃), 25.0 (s, C-3), 30.5 (s, C-4), 79.1 (C-5), 137.4 (C-2),172.9 (—C═O). IR (Neat film) 1676 (C═O), 1585 (C═N), 1215 (N—O). ESI-MScalcd for C₆H₁₀N₂O₂Na+ m/z 165.0634, found 165.0637 amu. Anal. Calcd.for C₆H₁₀N₂O₂: C, 50.81; H, 7.23; N, 19.14. Found: C, 50.61; H, 7.25; N,19.31.

2-Amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH₂-AMPO). NH₂-AMPOwas prepared using the procedure described previously³⁹ using cyanide ascatalyst for the aminolysis of esters. A solution of 100 mg (0.584 mmol)of EMPO in 25 mL of ca. 12N NH₃ in MeOH and 28 mg (0.058 mmol) of NaCNwas heated to 60° C. in a sealed tube for 40 hr. The solvent wasevaporated and the residue was redissolved in CH₂Cl₂. The organic phasewas extracted with minimal amount of water and dried over MgSO₄.Evaporation of the solvent gave mixture of AMPO and NH₂-AMPO. The crudeproduct was purified by column chromatography using silica gel andmethanol-ethyl acetate (30:70) as solvent. The product was furtherpurified twice by column chromatography using EtOH as solvent whichafforded NH₂-AMPO (5 mg), mp. >200 (dec). ¹H NMR (400 MHz, D₂O, 4.58ppm) δ 1.40 (3 H, s, C(5)Me), 1.90-2.06 and 2.22-2.35 (2 H, m, C(4)H),2.59-2.65 (2 H, m, C(3)H). IR (Neat film) 3346 and 1661 (N—H), 1682(C═O), 1627 (C═N), 1201 (N—O). ESI-MS calcd for C₆H₁₁N₃O₂Na⁺ m/z180.0749, found 180.0743 amu.

Miscellaneous Spin Trapping Studies Fenton reaction system. A 50 μL 0.1M phosphate buffer solution containing 30 mM AMPO, 1% H₂O₂ and 65 mMFeSO₄ was transferred to a 50 μL capillary tube and EPR spectrum of thehydroxyl adduct was recorded over 5 min time period.

Trapping of SO₃ ^(•−), CO₂ ^(•−) and CH₃.CHOH radicals. 50 μL 0.1 Mphosphate buffer solution containing 30 mM AMPO, 1% H₂O₂ and 100 mM ofthe respective radical source NaHCO₂, Na₂SO₃, or ethanol with 65 mMfreshly prepared FeSO₄. The mixture was then transferred to 50 μLcapillary tube and EPR spectrum of the adduct was recorded over a 5 mintime period.

Trapping of GS. and t-BuO. radicals. 50 μL 0.1 M phosphate buffersolution containing 30 mM AMPO and 100 mM GSSG or (CH₃)₃CO—OC(CH₃)₃. Themixture was then transferred to 50 μL capillary tube and the radicalswere generated by UV photolysis. EPR spectrum of the adduct was recordedover a 5 min time period.

Trapping of O₂ ^(•−). Typical O₂ ^(•−) trapping experiments utilized theriboflavin-light system as described in the Kinetics section. Analternative O₂ ^(•−) generating system used a solution of 0.4 mMxanthine and 0.5 unit/mL xanthine oxidase, or 10 nM PMA and 8×105neutrophil cells in 25 mM AMPO. Spectra were acquired over a period of15 min. TABLE 1 EPR parameters of simulated radical adducts of AMPO^(a)Diasiereomers Hyperfine coupling constants (G) Radicals Generatingsystem (%) a_(N) a_(H) ^(β) a_(H) ^(γ) O₂ HX/XO 80 13.0 10.8 20 13.112.5 1.75 •OH Fe²⁺—H₂O₂ 69 14.0 13.5 31 14.0 12.5 CO₂ ^(•-b)Fe²⁺—H₂O₂—NaHCO₂ 47 14.25 18.15 14.53 16.48 SO₃ ^(•-c) Fe²⁺—H₂O₂—Na₂SO₃46 13.47 15.93 54 13.47 14.67 CH_(3•CHOH) ^(d) Fe²⁺—H₂O₂—EtOH 100 14.821.4 (CH₃)₃CO•^(e) (CH₃)₃COOC(CH₃)₃— 56 14.19 13.64 uv 44 13.85 12.79GS•^(f) GSSG-uv 90 14.26 14.96 10 14.39 12.06^(a)Based on the simulation program by Rockenbauer, A., et al.35Simulated spectrum contains:^(b)19% C-centered adduct and 12% OH adduct^(c)23% C-centered adduct^(d)13% OH adduct^(e)10% C-centered adduct and 12% OOH-like adduct^(f)12% C-centered adduct

X-ray Crystallographic Data For AMPO The data collection crystal of AMPOwas a thin colorless plate. Examination of the diffraction pattern on aCCD diffractometer indicated a monoclinic crystal system. All work wasdone at 200 K. The data collection strategy was set up to measure aquadrant of reciprocal space with a redundancy factor of 3.1, whichmeans that 90% of the reflections were measured at least 3.1 times. Acombination of phi and omega scans with a frame width of 2.0° was used.Data integration was done with Denzo⁷ and scaling and merging of thedata was done with Scalepack⁷. Merging the data and averaging thesymmetry equivalent reflections resulted in an Rint value of 0.044.

The structure was solved by the direct methods in SHELXS-86⁸.Full-matrix least-squares refinements based on F² were performed inSHELXL-93⁹.

For the methyl group, the hydrogen atoms were added at calculatedpositions using a riding model with U(H)=1.5 * Ueq(bonded atom). Thetorsion angle, which defines the orientation of the methyl group aboutthe C—C bond, was refined. The two hydrogen atoms bonded to N(2) werefound on a difference map and then refined isotropically. The remaininghydrogen atoms were included in the model at calculated positions usinga riding model with U(H)=1.2 * Ueq(attached atom). The final refinementcycle was based on 1173 intensities and 100 variables and resulted inagreement factors of R1(F)=0.060 and wR2(F²=0.103). For the subset ofdata with I>2σ(I), the R1(F) value is 0.040 for 907 reflections. Thefinal difference electron density map contains maximum and minimum peakheights of 0.14 and −0.25 e/Å³. Neutral atom scattering factors wereused and include terms for anomalous dispersion¹⁰. The PLATON program¹¹was used to calculate the metrical parameters for the hydrogen bonds.TABLE 2 Crystallographic Data for AMPO. empirical formula C₆H₁₀N₂O₂formula weight 142.16 crystal system monoclinic space group. Z P2(1)/c,4 a (Å) 10.758(4) b (Å) 5.764(2) c (Å) 11.105(5) b (°) 104.670(10) unitcell volume Å³ 666.2(5) ρ_(calc) (g cm⁻³) 1.417 T(K) 200(2) K wavelength0.71073 Å μ(mm⁻¹) 0.108 final R^(a) R₁ =0.0401 wR₂ = 0.0950^(a)R₁= Σ∥F_(o)| - |F_(c) ∥/Σ|F_(o) | with [ > 2 (I) and wR₂ =[Σ[w(F_(o) ² -F_(c) ²)²]/Σ[w(F_(o) ²)²]]^(½).

X-ray Crystallographic Data For NH₂-AMPO The data collection crystalNH₂-AMPO was a pale yellow, approximately rectangular plate. Examinationof the diffraction pattern on a CCD diffractometer indicated anorthorhombic crystal system. All work was done at 200 K. The datacollection strategy was set up to measure an octant of reciprocal spacewith a redundancy factor of 4.4, which means that 90% of the reflectionswere measured at least 4.4 times. A combination of phi and omega scanswith a frame width of 2.0° was used. Data integration was done withDenzo,¹² and scaling and merging of the data was done with Scalepack.¹²Merging the data and averaging the symmetry equivalent reflectionsresulted in an Rint value of 0.044. The teXsan⁵⁶ package indicated thespace group to be P2₁2₁2₁.

The structure was solved by direct methods in SHELXS-86.¹³ Based on theX-ray data only, it is not possible to determine which enantiomer ispresent in this structure. Full-matrix least squares refinement based onF were performed in SHELXL-93.¹⁴

For the methyl group, the hydrogen atoms were added at calculatedpositions using a riding model with U(H)=1.5 * Ueq(bonded atom). Thetorsion angle, which defines the orientation of the methyl group aboutthe C—C bond, was refined. The hydrogen atoms bonded to nitrogen atomswere refined isotropically. The remaining hydrogen atoms were includedin the model at calculated positions using a riding model withU(H)=1.2 * Ueq(attached atom). The final refinement cycle was based on1709 intensities and 117 variables and resulted in agreement factors ofR1(F)=0.059 and wR2(F²=0.088). For the subset of data with I>2 σ(I), theR1(F) value is 0.040 for 1369 reflections. The final difference electrondensity map contains maximum and minimum peak heights of 0.16 and −0.17e/Å³. Neutral atom scattering factors were used and include terms foranomalous dispersion¹⁵. The PLATON program¹⁶ was used to calculate themetrical parameters for the hydrogen bonds.

All of the hydrogen atoms of the NH₂ groups are involved in intra andintermolecular hydrogen bonds. TABLE 3 Crystallographic Data forNH₂-AMPO. empirical formula C₆H₁₁N₃O₂ formula weight 157.18 crystalsystem orthorhombic space. group, Z P2₁ 2₁ 2₁, 4 a (Å) 7.582(2) b (Å)9.269(3) c (Å) 10.682(3) unit cell volume Å³ 750.7(4) ρ_(calc)(g cm³)1.391 T(K) 200(2) wavelength 0.71073 Å μ (mm⁻¹) 0.106 final R^(a) R₁ =0.0397 0.0808^(a)R₁ = Σ∥F_(o)| - |F_(c)∥/Σ| F_(o) |with [ > 2 σ(1) and wR₂ =[Σ[w(F_(o) ²-F_(c) ²)²]/Σ[w(F_(o) ²]]^(½).

Decay Kinetics In a typical decay kinetic study, 50 μL solutioncontaining 25 mM of the nitrone and 100 μM riboflavin was irradiated for3 min in the cavity. The lowest-field peak decay was monitored as afunction of time over a period of 2680 s after the light source wasturned off. All data were the average of 3 or more measurements. TABLE 4First order approximation half-lives of nitrone-superoxide adducts andpartition coefficient of the nitrone spin traps at pH 7.2 and 23° C.K_(p) (n-octanol/ Spin trap k_(l) /10⁻⁴s⁻¹ t_(1/2)/min^(a) ref.water)^(b) AMPO 14.0 ± 1.2  8.3 ± 0.7 this work 0.03 EMP0 11.6 ± 0.5 9.9 ± 0.4 this work 0.33 8.6, 8.0 14,36 24.6 ± 2.7  18   DEPMPO 7.5 ±0.7 15.5 ± 1.4  this work 0.16  8.13 14.2  16   DMPO 129.0  0.9  0.06^(a)Based on the first-order rate constant and values are mean averageof 3-6 measurements.^(b)Shaken for 2 hrs at 37° C.

These DMPO-type spin traps include the alkoxyphosphorylated nitrones5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO)¹⁷⁻¹⁹ and5-diisopropyloxyphosphoryl-5-methyl-1-pyrroline N-oxide (DIPPMPO)²⁰, andthe alkoxycarbonyl-nitrones, 5-ethoxycarbonyl-5-methyl-1-pyrrolineN-oxide (EMPO)²¹⁻²⁴ and 5-butoxycarbonyl-5-methyl-1-pyrroline N-oxide(BocMPO).²⁴⁻²⁷

The examples included herein are for illustration and are not meant tolimit the scope of the invention. All articles cited are incorporatedherein by reference.

REFERENCES

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1. A compound of formula I:

wherein R¹ is selected from the group consisting of halo, alkyl,substituted alkyl, branched alkyl, and cyclic alkyl, and R² is selectedfrom H and NH₂; or a salt thereof.
 2. The compound of claim 1 wherein R¹is alkyl or a salt thereof.
 3. The compound of claim 2 wherein R² is H;or a salt thereof.
 4. The compound of claim 2 wherein R² is NH₂; or asalt thereof.
 5. The compound of claim 2 wherein R² is methyl; or a saltthereof.
 6. The compound of claim 5 wherein R² is H; or a salt thereof.7. The compound of claim 5 wherein R² is NH₂; or a salt thereof.
 8. Thecompound of claim 1 wherein the compound is5-carbamoyl-5-methyl-1-pyrroline N-oxide (AMPO); or a salt thereof. 9.The compound of claim 1 wherein the compound is2-amino-5-carbamoyl-5-methyl-1-pyrroline N-oxide (NH₂-AMPO); or a saltthereof.
 10. A spin trap of formula I:

wherein R¹ is selected from the group consisting of halo, alkyl,substituted alkyl, branched alkyl, and cyclic alkyl, and R² is selectedfrom H and NH₂; or a salt thereof.
 11. The spin trap of claim 10 whereinR¹ is methyl and R² is H.
 12. The spin trap of claim 10 wherein R¹ ismethyl and R² is NH₂.
 13. A method of studying the formation of radicalsin an aqueous solution comprising the steps of: (a) contacting theradicals with a compound of formula I

 wherein R¹ is selected from the group consisting of halo, alkyl,substituted alkyl, branched alkyl, and cyclic alkyl, and R² is selectedfrom H and NH₂; or a salt thereof; and (b) detecting the radicalsspectroscopically.
 14. The method of claim 13 wherein the radicals aredetected using Electron Spin Resonance (ESR) spectroscopy.
 15. Themethod of claim 14 wherein the radicals are detected in vitro.
 16. Themethod of claim 14 wherein the radicals are detected in vivo.
 17. Themethod of claim 14 wherein the radicals studied are selected from thegroup consisting of hydroxyl, superoxide, C-centered, sulfite, andtert-butoxyl radicals, or combinations thereof.
 18. The method of claim13 wherein the radicals are detected using nuclear magnetic resonance(NMR).
 19. The method of claim 18 wherein the radicals are detected invitro.
 20. The method of claim 18 wherein the radicals are detected invivo.